Method and device for combined detection of viral and bacterial infections

ABSTRACT

A lateral flow assay detects and differentiates between viral and bacterial infections. A combined point of care diagnostic device tests markers for viral infection and markers for bacterial infection, to effectively assist in the rapid differentiation of viral and bacterial infections. In one preferred embodiment, the bacterial marker is CRP. In another preferred embodiment, the viral marker is MxA. In some embodiments, it is unnecessary to lyse the cells in the sample prior to applying it to the device.

REFERENCE TO RELATED APPLICATIONS

This application claims one or more inventions which were disclosed inProvisional Application No. 61/179,059, filed May 18, 2009, entitled“METHOD AND DEVICE FOR COMBINED DETECTION OF VIRAL AND BACTERIALINFECTIONS”. The benefit under 35 USC §119(e) of the United Statesprovisional application is hereby claimed, and the aforementionedapplication is hereby incorporated herein by reference.

This application is also a continuation-in-part application ofapplication Ser. No. 12/469,207, filed May 20, 2009, entitled“NANOPARTICLES IN DIAGNOSTIC TESTS”, which claimed priority fromProvisional Application No. 61/071,833, filed May 20, 2008, entitled“NANOPARTICLES IN DIAGNOSTIC TESTS”, application Ser. No. 12/481,631,filed Jun. 10, 2009, entitled “COMBINED VISUAL/FLUORESCENCE ANALYTEDETECTION TEST”, which claimed priority from Provisional Application No.61/060,258, filed Jun. 10, 2008, entitled “COMBINED VISUAL/FLUORESCENCEANALYTE DETECTION TEST”, application Ser. No. 12/502,626, filed Jul. 14,2009, entitled “LATERAL FLOW NUCLEIC ACID DETECTOR”, which claimedpriority from Provisional Application No. 61/080,879, filed Jul. 15,2008, entitled “LATERAL FLOW NUCLEIC ACID DETECTOR” and application Ser.No. 12/502,662, filed Jul. 14, 2009, entitled “IN SITU LYSIS OF CELLS INLATERAL FLOW IMMUNOASSAYS”, which claimed priority from ProvisionalApplication No. 61/098,935, filed Sep. 22, 2008, entitled “IN SITU LYSISOF CELLS IN LATERAL FLOW IMMUNOASSAYS” and PCT application Serial NumberPCT/US2009/057775, filed Sep. 22, 2009, entitled “METHOD AND DEVICE FORCOMBINED DETECTION OF VIRAL AND BACTERIAL INFECTIONS”. Theaforementioned applications are hereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

The invention pertains to the field of lateral flow immunoassays. Moreparticularly, the invention pertains to a lateral flow immunoassay thatrapidly detects viral and bacterial infection.

Description of Related Art

Fever is a common cause of childhood visits to urgent care centers forboth family practice and pediatric offices. Most commonly, this relatesto either a respiratory infection or gastroenteritis. The high incidenceof fever in children and the precautious administration of unnecessaryantibiotics is reason to develop a rapid screening test for thebiomarkers that indicate viral and/or bacterial infection.

It is often challenging to differentiate viral from bacterialinfections. This is especially true in young children that cannotverbalize their symptoms and in the outpatient setting where access tolaboratory diagnostics is expensive, time consuming, and requiresseveral days to produce a result. More recently, many new diagnosticmarkers have been identified. Several of these markers show greatpromise to differentiate viral from bacterial infections. Two suchproteins include MxA and C-Reactive Protein (CRP). Most respiratoryinfections are related to pharyngitis of which 40% are caused by virusesand 25-50% by group A beta hemolytic streptococcus. The lesser causesare acute bronchiolitis and pneumonia.

Severe community-acquired pneumonia is caused by bacterial infections inaround 60% of cases, requiring admission to an intensive care unit (ICU)for about 10% of patients. The remaining 30% are related to respiratoryviruses.

About 80% of all antimicrobials are prescribed in primary care, and upto 80% of these are for respiratory tract indications. Respiratory tractinfections are by far the most common cause of cough in primary care.Broad spectrum antibiotics are often prescribed for cough, includingacute bronchitis, and many of these prescriptions will benefit patientsonly marginally if at all, and may cause side effects and promoteantibiotic resistance. Factors that urge physicians to give antibioticsinclude the absence of an adequate diagnostic marker of bacterialinfections, the concern about lack of patient follow-up, and the timepressure.

Mx proteins are members of the superfamily of high molecular weightGTPases. Accordingly, these GTPases are upregulated by type I alpha/betaor type II interferons (IFN). The Mx GTPases are expressed exclusivelyin IFN alpha/beta but not IFN gamma treated cells. Type I interferonsplay important roles in innate immune responses and haveimmunomodulatory, antiproliferative, and antiviral functions. Human MxA,a 78 kDa protein, accumulates in the cytoplasm of IFN treated cells andinhibits the replication of a wide range of viruses. MxA protein mayoffer certain advantages as a marker for viral infection over the otherinduced proteins such as 2′, 5′-oligoadenylate synthetase, because ofits lower basal concentration, longer half-life (2.3 days) and fastinduction. MxA mRNA is detectable in isolated peripheral blood whiteblood cells stimulated with IFN within 1 to 2 h of IFN induction, andMxA protein begins to accumulate shortly thereafter.

Studies have shown that MxA protein expression in peripheral blood is asensitive and specific marker for viral infection. The higher MxA levelsin the viral infection group compared with the bacterial infection groupcan be explained by the fact that the MxA protein is induced exclusivelyby type I IFN and not by IFN-gamma, IL-1, TNF-alpha, or any of the othercyotokines by bacterial infection. Serum type I IFN levels remain withinnormal limits, even in patients with severe bacterial infections.

Similarly, most viral infections have been reported to cause littleacute phase response, and low C-Reactive Protein (CRP) concentrationshave been used to distinguish illnesses of viral origin from those ofbacterial etiology. Because the plasma concentration of CRP increasesrapidly after stimulation and decreases rapidly with a short half-life,CRP can be a very useful tool in diagnosing and monitoring infectionsand inflammatory diseases. In Scandinavia, point of care CRP testing ispart of the routine evaluation of patients with respiratory infectionsin general practice, and its use has proved cost-effective. In generalpractice, CRP is found valuable in the diagnosis of bacterial diseasesand in the differentiation between bacterial and viral infections. Oftenthe diagnostic value of CRP is found superior to that of the erythrocytesedimentation rate (ESR) and superior or equal to that of the whiteblood cell count (WBC).

Clinically, it can be challenging to differentiate certain systemicviral and bacterial infections. Bacterial cultures are usually performedin cases of severe infection such as pneumonia, or when the consequenceof missing a diagnosis can lead to severe complications, such as withStrep throat. Often times, cultures are difficult to obtain.Unfortunately, viral cultures are not routinely performed due to thesignificant time delay in receiving results. New viral screening PCRpanels are useful but they are expensive and do not provide informationat the point of care. Thus, there remains a need for a simple, easy touse diagnostic test that is capable of differentiating viral andbacterial infections.

SUMMARY OF THE INVENTION

The present invention provides a lateral flow assay that is capable ofdetecting and differentiating viral and bacterial infections. A combinedpoint of care diagnostic device tests markers for viral infection andmarkers for bacterial infection, to effectively assist in the rapiddifferentiation of viral and bacterial infections. In one preferredembodiment, the bacterial marker is CRP. In another preferredembodiment, the viral marker is MxA. In some embodiments of theinvention, it is unnecessary to lyse the cells in the sample prior toapplying it to the device.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows rapid screening test window visual test results todistinguish viral and bacterial infections and an interpretation ofthose results.

FIG. 2 shows three cassettes with different colored test lines.

FIG. 3 shows a comparison of a two line detector, where both lines arethe same color, and an extra sensitive two line detector, where the twolines are different colors.

FIG. 4A shows a device with a test line corresponding to the presence ofa viral marker and a second, separate test line that detects thepresence of a bacterial marker in an embodiment of the presentinvention.

FIG. 4B shows a device with a test line corresponding to the presence ofa viral marker and a second, separate test line that detects thepresence of a bacterial marker in another embodiment of the presentinvention.

FIG. 5A shows a sample analysis device including a lysis zone locatedbetween a sample application zone and a reagent zone in an embodiment ofthe present invention.

FIG. 5B shows a sample analysis device including a lysis zoneoverlapping a sample application zone in an embodiment of the presentinvention.

FIG. 5C shows a sample analysis device including a lysis zoneoverlapping a reagent zone in an embodiment of the present invention.

FIG. 5D shows a sample analysis device including a lysis zoneoverlapping a sample application zone and a reagent zone in anembodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a lateral flow assay that is capable ofdifferentiating viral and bacterial infections. A combined point of carediagnostic device tests markers for both viral and bacterial infectionand can effectively assist in the rapid differentiation of viral andbacterial infections, for example at the outpatient office or during anurgent care visit. This ability can dramatically reduce health carecosts by limiting misdiagnosis and the subsequent overuse ofantibiotics. Such a practice may limit antibiotic allergies, adverseevents, and antibiotic resistance. The rapid result obtained from thetest also permits a diagnosis while the patient is still being examinedby the practitioner. In a preferred embodiment, the test result isobtained in under 10 minutes after applying the sample to the device,and it is preferably read at approximately 10 minutes. In samples thatare highly positive, the test line is visible within approximately 1-5minutes.

In a preferred embodiment of the present invention, the lateral flowimmunoassay device of the present invention includes asample-transporting liquid, which can be a buffer, and a chromatographytest strip containing one or several fleece materials or membranes withcapillary properties through which sample flows. Some preferredmaterials and membranes for the test strip include, but are not limitedto, Polyethylene terephthalate (PET) fibers, such as Dacron® fibers,nitrocellulose, polyester, nylon, cellulose acetate, polypropylene,glass fibers, and combinations of these materials and their backings. Insome embodiments of the invention, it is unnecessary to lyse the cellsin the sample prior to applying it to the test strip.

One preferred method of the present invention uses a sample analysisdevice, for example a chromatography test strip, to determine if aninfection is bacterial or viral. In this method, a sample is collected,and transferred to the chromatography test strip. In a preferredembodiment, the sample is a sample including leukocytes. The test stripincludes a reagent zone. The reagent zone preferably includes at leastone first reagent specific to a bacterial marker such that, when thebacterial marker present in the sample contacts the first reagent, afirst labeled complex forms. The reagent zone also preferably includesat least one second reagent specific to a viral marker such that, whenthe viral marker present in the sample contacts the second reagent, asecond labeled complex forms. A detection zone includes both a bacterialmarker binding partner which binds to the first labeled complex and aviral marker binding partner which binds to the second labeled complex.The sample is then analyzed for the presence of the viral marker and/orthe bacterial marker.

A preferred embodiment of a device of the present invention includes asample application zone. The device also includes a reagent zone, whichincludes at least one first reagent specific to a bacterial marker suchthat, when a bacterial marker present in the sample contacts the firstreagent, a first labeled complex forms and at least one second reagentspecific to a viral marker such that, when a viral marker present in thesample contacts the second reagent, a second labeled complex forms. Adetection zone on the device includes a bacterial marker binding partnerwhich binds to the first labeled complex and a viral marker bindingpartner which binds to the second labeled complex. One example of adevice that could be used is a chromatography test strip.

In a preferred embodiment, the presence of the viral marker or thebacterial marker is indicated by a test line visible to the naked eye.The presence of the viral marker may be indicated by a first test linewhile the presence of the bacterial marker is indicated by a second testline. In some embodiments, the first test line displays a first colorwhen positive and the second test line displays a second color differentfrom the first color when positive. In embodiments where both the firsttest line and the second test line are located in the same space on thesample analysis device, a third color is preferably formed when both thefirst test line and the second test line are positive. In otherembodiments, the two test lines are spatially separate from each otheron the device.

In one preferred embodiment, the bacterial marker is CRP. In anotherpreferred embodiment, the viral marker is MxA. In some preferredembodiments, the detection zone also includes a control line that isvisible to the naked eye when the device is working.

In one preferred embodiment, the marker for viral infection is MxA andthe marker for bacterial infection is C-reactive protein (CRP). High MxAprotein levels are strongly correlated with systemic viral infection andincreased CRP is more associated with bacterial infections. The presentinvention includes a rapid infectious screening test for identifying MxAand CRP in samples. MxA is present in leukocytes (white blood cells).Therefore, the sample can be taken anywhere leukocytes are available,for example in a peripheral blood sample, nasopharyngeal aspirates,tears, spinal fluid, and middle ear aspirates.

In some preferred embodiments, the threshold concentration of CRP in asample needed to elicit a positive result is approximately 15 mg/L. Inother preferred embodiments, the threshold concentration of MxA in asample to elicit a positive result may be as low as approximately 15ng/ml; however, the threshold concentration may by higher, in a rangefrom approximately 20 ng/ml to approximately 250 ng/ml. The thresholdconcentration may depend on the size of the sample being applied to thetest strip, as well as its dilution, if applicable.

In other embodiments, other markers for viral infection and/or bacterialinfection may be used. For example, approximately 12% of host genesalter their expression after Lymphocytic Choriomeningitis Virus (LCMV)infection, and a subset of these genes can discriminate between virulentand nonvirulent LCMV infection. Major transcription changes have beengiven preliminary confirmation by quantitative PCR and protein studiesand are potentially valuable candidates as biomarkers for arenavirusdisease. Other markers for bacterial infection include, but are notlimited to, procalcitonin, urinary trypsin inhibitor (uTi),lipopolysaccharide, IL-1, IL-6, IL-8, IL-10, ESR and an elevated WBCcount (increased bands), Lactate, Troponin, vascular endothelial growthfactor, platelet derived growth factor, cortisol, proadrenomedullin,macrophage migratory inhibitory marker, activated protein C, CD 4, 8,13, 14, or 64, caspase, placenta derived growth factor, calcitoningene-related peptide, high mobility group 1, copeptin, naturieticpeptides, lipopolysaccharide binding protein, tumor necrosis factoralpha, circulating endothelial progenitor cells, complement 3a, andtriggering receptor expressed on myeloid cells (trem-1).

In one embodiment, the infections being distinguished are respiratoryinfections. In other embodiments, other types of infections, which canbe bacterial or viral, are differentiated using the system of thepresent invention. Some examples include, but are not limited to,encephalitis, meningitis, gastroenteritis, febrile respiratory illness(including bronchitis, pharyngitis, pneumonia), sinusitis, otitis media,urinary tract infections, and conjunctivitis.

Lateral flow devices are known, and are described in, e.g., U.S.Published Patent Application Nos. 2005/0175992 and 2007/0059682. Thecontents of both of these applications are incorporated herein byreference. Other lateral flow devices known in the art couldalternatively be used with the systems and methods of the presentinvention.

U.S. Published Patent Application No. 2007/0059682 discloses detectingan analyte and a sample which can also contain one or more interferingsubstances. This publication teaches separating the analyte from theinterfering substances by capturing the interfering substances on thechromatographic carrier, and detecting the analyte on the carrierseparated from the interfering substances.

U.S. Published Patent Application No. 2005/0175992 discloses a methodfor detecting targets, such as pathogens and/or allergy-associatedcomponents, in a human body fluid where the body fluid sample iscollected by a collection device, such as a swab member. The samples aretransferred from the swab member to a sample analysis device, on whichan analysis of the targets can occur by immunochemical or enzymaticmeans. The test result is capable of being displayed within a very shortperiod of time and can be directly read out by the user. This enablespoint-of-care testing with results available during a patient visit. Theinventions disclosed in this copending application are particularlyadvantageous for the diagnosis of conjunctivitis.

In a method of the invention, the sample to be analyzed is applied to achromatographic carrier. The carrier can be made of one singlechromatographic material, or preferably several capillary activematerials made of the same or different materials and fixed on a carrierbacking. These materials are in close contact with each other so as toform a transport path along which a liquid driven by capillary forcesflows from an application zone, passing a reagent zone, towards one ormore detection zones and optionally a waste zone at the other end of thecarrier. In other embodiments, the liquid passes the reagent zone priorto flowing into the sample application zone. In an especially preferredembodiment, the carrier is a chromatographic test strip.

In some embodiments, the sample is directly applied to the carrier bydipping the carrier's application zone into the sample. Alternatively,application of the sample to the carrier may be carried out bycollecting the sample with a dry or wetted wiping element from which thesample can be transferred, optionally after moistening, to the carrier'sapplication zone. Usually, the wiping element is sterile and may be dryor pretreated with a fluid before the collection step. Materialssuitable for wiping elements according to the invention may comprisesynthetic materials, woven fabrics or fibrous webs. Some examples ofsuch wiping elements are described in German Patents DE 44 39 429 and DE196 22 503, which are hereby incorporated by reference.

Depending on the type of detection method, different reagents arepresent in the carrier's reagent zone, which, in some embodiments, ispreferably located between the application zone and the detection zoneor, in other embodiments, is preferably located before the applicationzone. In a sandwich immunoassay, it is preferred to have a labeled,non-immobilized reagent in the reagent zone that is specific to eachbacterial and viral marker that is being detected. Thus, when a viral orbacterial marker present in the sample contacts the correspondinglabeled viral or bacterial reagent present in the reagent zone, alabeled complex is formed between the marker and the correspondinglabeled reagent. The labeled complex in turn is capable of forming afurther complex with an immobilized viral or bacterial marker bindingpartner at a test line in the detection zone. In a competitiveimmunoassay, the reagent zone preferably contains a labeled,non-immobilized marker analogue which competes with the marker for theimmobilized marker binding partner in the detection zone. The markerbinding partners in the reagent zone and in the detection zone arepreferably monoclonal, polyclonal or recombinant antibodies or fragmentsof antibodies capable of specific binding to the corresponding marker.

In a preferred embodiment, the present invention provides for thereduction of interfering substances that might be present in the sampleto be tested. Since an interfering substance, e.g. a human anti-mouseantibody (HAMA), may also be capable of forming a complex with thelabeled, non-immobilized reagent of the reagent zone and the immobilizedbinding partner of the detection zone, thus indicating a positive testresult in the immunoassay, the carrier may further include at least onecapturing zone. Each capturing zone contains an immobilized capturingreagent specifically binding to a certain interfering substance, therebyimmobilizing the interfering substance in the capturing zone. As thecapturing zone is separated from the detection zone by space, and thesample starts to migrate over the reagent zone and the capturing zonebefore reaching the carrier's detection zone, the method allows aseparation of the interfering substance or substances from the analyteor analytes of interest. Preferably, the capturing zone is locatedbetween the reagent zone and the detection zone. However, the capturingzone may also be located between the application zone and the reagentzone.

Detection of the marker may be achieved in the detection zone. Thebinding molecule immobilizes the labeled complex or the labeledmarker-analogue by immune reaction or other reaction in the detectionzone, thus building up a visible test line in the detection zone duringthe process. Preferably, the label is an optically detectable label.Forming a complex at the test line concentrates and immobilizes thelabel and the test line becomes visible for the naked eye, indicating apositive test result. Particularly preferred are direct labels, and moreparticularly gold labels which can be best recognized by the naked eye.Additionally, an electronic read out device (e.g. on the basis of aphotometrical, acoustic, impedimetrical, potentiometric and/oramperometric transducer) can be used to obtain more precise results anda semi-quantification of the analyte. Other labels may be latex,fluorophores or phosphorophores.

In one embodiment, the sensitivity of visually read lateral flowimmunoassay tests is enhanced by adding a small quantity of fluorescingdye or fluorescing latex bead conjugates to the initial conjugatematerial. When the visible spectrum test line is visibly present, thetest result is observed and recorded. However, in the case of weakpositives that do not give rise to a distinct visual test line, a lightof an appropriate spectrum, such as a UV spectrum, is cast on the testline to excite and fluorescent the fluorescing latex beads which arebound in the test line to enhance the visible color at the test line.

In a preferred embodiment, the reagents are configured such that thevisible test line corresponding to the presence of the viral marker willbe separate from the test line corresponding to the presence of thebacterial marker. Therefore, it can be readily determined whether thesample contained bacterial or viral markers (or both) simply by thelocation of the development of the test lines in the detection zone. Inanother preferred embodiment, the reagents may be chosen such thatdifferently colored test lines are developed. That is, the presence of aviral marker will cause the development of a differently colored linethan that developed by the presence of a bacterial marker. For example,the label corresponding to the reagent recognizing the viral marker maybe red, whereas the label corresponding to the reagent recognizing thebacterial marker may be green. Differently colored labels that may beattached to the non-immobilized reagents are well known. Some examplesinclude, but are not limited to, colloidal gold, colloidal selenium,colloidal carbon, latex beads, paramagnetic beads, fluorescent andchemiluminescent labels and mixtures thereof.

FIGS. 4A and 4B show a chromatography test strip (400) with a test line(402) corresponding to the presence of a viral marker and a second,separate test line (403) that detects the presence of a bacterialmarker. The sample is applied to the application zone (401) of thechromatography test strip (400). As shown in FIG. 4A, the sample thenpasses a reagent zone (460) containing at least one labeled viralbinding partner and at least one labeled bacterial binding partner thatis eluted by and then able to migrate with a sample transport liquid(e.g. a buffer solution). Alternatively, as shown in FIG. 4B, thereagent zone (460) is located upstream of the sample application zone(401) such that the labeled binding partners in the reagent zone areeluted by the sample transport liquid and travel to the sample. Thelabeled viral binding partner is capable of specifically binding to aviral marker of interest to form a complex which in turn is capable ofspecifically binding to another specific reagent or binding partner inthe detection zone. The labeled bacterial binding partner is capable ofspecifically binding to a bacterial marker of interest to form a complexwhich in turn is capable of specifically binding to another specificreagent or binding partner in the detection zone. Although not shown inthese Figures, an absorbent pad, as well as other known lateral flowimmunoassay components including, but not limited to, a waste zone, acarrier backing, a housing, and an opening in the housing for resultread out, may optionally also be a component of the test strip (400) inthese embodiments.

The test strip (400) also includes a detection zone (405) containing atleast one first section for detection of a viral marker, e.g. a testline (402), including an immobilized specific binding partner,complementary to the viral reagent complex formed by the viral markerand its labeled binding partner. Thus, at the test line (402), detectionzone binding partners trap the labeled viral binding partners from thereagent zone (460) along with their bound viral markers. Thislocalization of the viral marker with its labeled binding partners givesrise to an indication at the test line (402). At the test line (402),the presence of the viral marker is determined by qualitative and/orquantitative readout of the test line (402) indication resulting fromthe accumulation of labeled binding partners.

The detection zone (405) also includes at least one second section fordetection of a bacterial marker, e.g. a test line (403), including animmobilized specific binding partner, complementary to the bacterialreagent complex formed by the bacterial marker and its labeled bindingpartner. Thus, at the test line (403), detection zone binding partnerstrap the labeled bacterial binding partners from the reagent zone (460)along with their bound bacterial markers. This localization of thebacterial marker with its labeled binding partners gives rise to anindication at the test line (403). At the test line (403), the presenceof the bacterial marker is determined by qualitative and/or quantitativereadout of the test line (403) indication resulting from theaccumulation of labeled binding partners. While test line (402) isupstream of test line (403) relative to the direction of flow (408) inthe figures, in alternative embodiments, test line (403) is upstream oftest line (402). In still other embodiments, test lines (402) and (403)are located in the same location on the test strip.

Optionally, the detection zone (405) may contain further test lines todetect other viral and/or bacterial markers, as well as a control line(404). The control line (404) indicates that the labeled specificbinding partner traveled through the length of the assay, even though itmay not have bound any viral or bacterial markers, thus confirmingproper operation of the assay. As shown in FIGS. 4A through 4B, thecontrol zone (404) is preferably downstream of the test lines (402) and(403). However, in other embodiments, the control zone (404) may belocated upstream of either or both of the test lines (402) and (403).

In a preferred embodiment, the control line (404) includes an antibodyor other recombinant protein which binds to a component of the elutionmedium or other composition being used in the test. In embodiments wherenucleic acids are the targets, the control line (404) preferablyincludes a nucleic acid complementary to the labeled nucleic acid beingused as a binding partner for the target nucleic acid.

Although only one test line is shown in the figures for each of theviral and bacterial markers, multiple test lines for both or either ofthe viral and bacterial markers may be used within the spirit of theinvention. In some embodiments where there are multiple bacterial and/orviral targets, the presence of each target preferably corresponds to aseparate test line (402) or (403). In other embodiments, both thebacterial marker and the viral marker are detected on a single testline. In these embodiments, the presence of both a bacterial marker anda viral marker on the same test line has different characteristics thanthe presence of either a bacterial or viral marker alone. For example,the presence of both a bacterial marker and a viral marker on the sametest line may be visually indicated by a different color than thepresence of either a bacterial marker or a viral marker alone.

Fresh whole blood samples of patients showing symptoms of viralinfections (flu like symptoms and fever of >100.5° F.)) were tested todetermine what levels of MxA in the blood could be detected with thelateral flow tests described herein. The lateral flow assays used inthese experiments had a similar configuration as the device shown inFIG. 4B described above, without a second test line for the presence ofa bacterial marker. More specifically, the test strip included a reagentzone upstream of a sample application zone. The reagent zone includedmobilizable antibodies to MxA (Kyowa Hakko Kirin Co., Ltd., Tokyo,Japan) labeled with colloidal gold. The test strip also included a testline in a detection zone. The test line included an immobilized antibodyfor MxA (Kyowa Hakko Kirin Co., Ltd., Tokyo, Japan). The control line inthe detection zone included rabbit anti-chicken antibody plus rabbit Ig(for an extra stabilizing effect), which binds to mobilized chicken IgYlabeled with blue latex beads.

The whole blood samples were collected with EDTA as the anticoagulant.In these tests, the amount of MxA protein in the blood samples wasdetermined using an MxA Protein ELISA Test kit (Kyowa Hakko Kirin Co.,Ltd., Tokyo, Japan). The blood was lysed 1:10 with lysing solutionprovided in the kit, prior to being applied to the test strip. 100 μl oflysed blood was tested in the ELISA test. 10 μl of lysed blood was usedas the sample in the MxA lateral flow test.

The lysed blood samples were applied to the application zone of the teststrip. The labeled MxA antibodies in the reagent were eluted by thesample transport liquid and traveled to the blood samples. At the testline, the immobilized MxA antibody trapped any labeled MxA antibody fromthe reagent zone bound to MxA. This localization of the MxA with itslabeled antibody gave rise to a red visual indication at the test lineif there was a sufficient concentration of MxA.

TABLE 1 Calibrator Lateral Concentration Flow MxA (ng/ml) OD Test 242.223 + 12 1.259 Shadow 6 0.700 Not tested 3 0.391 Not tested 1.5 0.220Not tested .75 0.140 Not tested 0.38 0.102 Not tested

Table 1 shows the MxA ELISA kit standards run per the test instructions.As shown in Table 1, an MxA concentration of 24 ng/ml produced apositive result in the lateral flow test. The kit standard was used togenerate the standard curve from which the MxA concentrations weredetermined.

Table 2 shows the results of clinical fresh whole blood samples ofpatients showing symptoms of viral infections (flu like symptoms andfever of >100.5° F.)).

TABLE 2 Concentration × Lateral Concentration dilution Flow MxA SampleOD (ng/ml) (10×) (ng/ml) Test A 0.008 0 0 − B 0.123 0.591 5.911 − C1.125 10.489 104.894 + D 0.111 0.487 4.872 − E 0.068 0.121 1.211 − F0.300 2.177 21.77 + G 0.027 0 0 −

The OD (optical density) values were used in combination with thestandard curve from the kit's standard in order to determine the MxAconcentration in the samples. The concentration (ng/ml) column was theconcentration as diluted with the lysing agent. Theconcentration×dilution (10×) (ng/ml) column was the actual concentrationin the whole blood sample. As shown in the table, the lateral flow testproduced a positive result for MxA in samples C and F, which hadapproximately 105 ng/ml of MxA and approximately 22 ng/ml of MxA,respectively, in the samples.

Table 3 shows the results of frozen whole blood samples from normalindividuals from the Tennessee blood bank. None of the blood samples hadany discernible amounts of MxA, and all of them were negative in thelateral flow test.

TABLE 3 CONCEN- CONCENTRATION × Lateral TRATION DILUTION Flow MxA SampleOD (ng/ml) (10×) (ng/ml) Test 1 0.003 0 0 − 2 0.014 0 0 − 3 0.008 0 0 −4 0.035 0 0 − 5 0.011 0 0 − 6 0.007 0 0 − 7 (0.017) 0 0 − 8 (0.006) 0 0− 9 0.028 0 0 − 10 0.012 0 0 − 11 0.028 0 0 − 12 0.02  0 0 − 13 0.044 00 − 14 0.023 0 0 − 15 0.032 0 0 − 16 0.02  0 0 − 17 0.032 0 0 − 18 0.0090 0 − 19 0.035 0 0 − 20 0.022 0 0 − 21 0.017 0 0 − 22 0.188 1.163 11.631− 23 0.014 0 0 − 24 0.005 0 0 − 25 0.039 0 0 −

Table 4 shows freshly frozen whole blood samples from BioReclamation(BioReclamation, Hicksville, N.Y.) of patients showing symptoms of viralinfections (flu like symptoms and fever of >100.5° F.)). None of thesepatients had ODs that corresponded to MxA levels higher thanapproximately 8 ng/ml. These samples were all negative in the lateralflow test.

TABLE 4 Concentration × Lateral Concentration dilution Flow MxA SampleOD (ng/ml) (10×) (ng/ml) Test 26 0.029 0 0 − 27 0.026 0 0 − 28 0.018 0 0− 29 0.146 0.792 7.92 − 30 0.004 0 0 − 31 0.128 0.635 6.35 −

The results of these tests indicate that the lateral flow testsdescribed herein can detect MxA levels at least as low as approximately20 ng/ml in a 10 μl sample (diluted 1:10).

One example of a rapid screening test for distinguishing viral andbacterial infection is shown in FIG. 1. As discussed above, MxA is adiagnostic marker for viral infection, while CRP is a diagnostic markerfor bacterial infection. In this example, a blue line (“control line” inA-D of the Figure) represents the control. A green line represents aC-reactive protein (CRP) level >15 mg/L (“CRP test” in A-D of thefigure). A red line represents an MxA level >20 ng/ml (“MxA test” in A-Dof the figure). A positive result for the MxA protein, with a negativeresult for the CRP protein indicates only a viral infection (Visual TestResult A). A positive result for the (CRP) with a negative result forthe MxA protein indicates only a bacterial infection (Visual Test ResultB). A positive result for both MxA and CRP indicates co-infection(infection with both a bacteria and a virus) (Visual Test Result C). Nobacterial or viral infection is indicated by a negative result for bothMxA and CRP (Visual Test Result D). While particular color lines arediscussed in this example, other colors, or the same colors at differentlocations on the test strip to indicate viral or bacterial markers, arewithin the spirit of the present invention.

When development of different colored lines is utilized, the lines mayor may not be separated by space. In the latter instance, the labels arechosen such that the color seen when both markers are present isdifferent from the colors seen when the individual markers are present.For example, the presence of the viral marker may be indicated by a redline; the presence of the bacterial marker by a blue line; and thepresence of both by a purple line (combined red and blue).

The use of two colors to distinguish acute and chronic infection isshown in FIG. 2. In the first cassette, only IgM antibodies are present,which indicates an acute infection. In this cassette, the test line isred. In the second cassette, the test line is blue because theimmunoglobulins are IgG. The third cassette shows an intermediate case,where both IgM and IgG antibodies are present. Consequently, the testline is purple. While this example is shown to test for IgMs and IgGs,the same concept is alternatively used with a single line which detectsboth viral and bacterial markers for infection.

In another preferred embodiment, the test strip may also include acontrol section which indicates the functionality of the test strip.FIG. 1 shows a control line. FIG. 2 shows an example where there is acontrol section for all three cassettes. If present, the control sectioncan be designed to convey a signal to the user that the device hasworked. For example, the control section may contain a reagent (e.g., anantibody) that will bind to the labeled reagents from the reagent zone.For example, an anti-mouse antibody may be used if the labeled antibodyis of murine origin, to confirm that the sample has permeated the teststrip. Alternatively, the control section may contain an anhydrousreagent that, when moistened, produces a color change or colorformation, e.g. anhydrous copper sulphate which will turn blue whenmoistened by an aqueous sample. As a further alternative, the controlsection could contain immobilized viral and bacterial markers which willreact with excess labeled reagent from the reagent zone. The controlsection may be located upstream or downstream from the detection zone. Apositive control indicator tells the user that the sample has permeatedthe required distance through the test device.

FIG. 3 compares two test strips, the “Adeno 1” and the “Adeno HS”, whichboth include control lines. In the Adeno 1, both the control (upper lineon each cassette) and test (lower line on each cassette) lines are red.In the Adeno HS, the control line is blue and the test line is red. Inembodiments where the control line is a different color than the testline, it is easier to distinguish between the two lines, and to ensurethat the test is working.

In some preferred embodiments, the devices and methods of the presentinvention include a lysis zone to help differentiate viral and bacterialinfections. In these embodiments, the sample that has been collected isnot lysed prior to collection and transfer to the sample analysisdevice. This decreases the number of steps needed to collect and preparethe sample for analysis. One situation where a lysis agent improvesassay efficiency is in assaying for the presence of MxA. As discussedherein, the presence of this protein can help to distinguish betweenbacterial and viral infection in febrile children. In situ lysis using acombination of 1% to 6% weight/volume CHAPS and 0.5% to 2% weight/volumeNP40 as the lysis agent improves detection of MxA in fresh or frozenwhole blood.

In the embodiments utilizing a lysis agent, following sample loading,the sample traveling with the transport liquid (buffer) will encounterthe lysis agent. The lysis agent will have preferably been pre-loadedonto the test strip and is eluted by the transport liquid. In somepreferred embodiments the lysis agent has been dried into the teststrip. Alternatively, the lysis agent may be pre-dried by freeze dryingor lyophilizing and then pre-loaded into the test strip. In otherembodiments, the lysis agent may be absorbed, adsorbed, embedded ortrapped on the test strip. The initially dried lysis agent is preferablylocalized between the sample application zone and a reagent zone. Inembodiments where the reagent zone is upstream of the sample applicationzone, the lysis zone is downstream of the sample application zone. Thelysing agent is preferably soluble in the sample transport liquid, andthe lysing agent is solubilized and activated upon contact with thesample transport liquid. The sample transport liquid then contains bothlysing agent in solution or suspension and sample components insuspension. Any lysis-susceptible components in a sample, then beingexposed in suspension to the lysing agent, are themselves lysed in situ.The running buffer then carries the analyte, including any lysis-freedcomponents, to the detection zone.

The location where the lysis agent is pre-loaded and dried can be variedas needed. In order to maximize the time that the sample has to interactwith the lysis agent as well as to minimize the amount of lysis agentreaching the detection zone, the dried, absorbed, adsorbed, embedded, ortrapped lysis agent may be located in or just downstream of the sampleapplication zone. Or, in order to minimize the distance along which thelysis product must travel before reaching the reagent zone, the driedlysis agent may be located closer to the reagent zone. In otherembodiments, the lysis agent may be included in the running buffer.

The concentration of lysis agent pre-loaded onto a test strip ispreferably between 0.001% and 5% weight/volume. The volume to bepre-loaded depends on where the lysis agent is pre-loaded. Appropriateranges are 1 to 10 microliters when pre-loaded into the sample collectorfleece (the sample application zone) or 5 to 50 microliters whenpre-loaded into the absorbent pad or into other locations within thetest strip. Ideally, the amount pre-loaded should be approximately 3microliters pre-loaded into the sample collector fleece or approximately10 microliters pre-loaded into the absorbent pad or into other locationswithin the test strip.

Selection of a specific lysing environment and agent will depend on theviral and bacterial markers and the assay. The pH and ionic strength arekey to the lysing environment. As to pH established by the lysis agent,a pH below 4.0 tends to precipitate materials, especially proteins.Higher pH, above approximately 10.0, tends to lyse materials such asproteins and cells walls. Therefore, a pH of approximately 10.0 or aboveis preferable for many applications. Alternatively, lower pH may bepreferred for nucleic acid targets.

As to ionic strength established by the lysis agent, both the high andlow ionic strength may be used to lyse. For example, a lower ionicstrength (hypotonic) tends to break up erythrocytes. For example, waterby itself can lyse erythrocytes. Higher ionic strength environments maybe used to rupture certain cell walls and membranes.

As to specific lysis agents, they may be grouped and selected based ontheir properties: salts, amphoteric and cationic agents, ionic andnon-ionic detergents. The salt, Ammonium Chloride (NH₄Cl), lyseserythrocytes. Other salts, including, but not limited to, highconcentrations of Sodium Chloride (NaCl) and Potassium Chloride (KCl),may rupture certain cell walls and membranes. Other lysis agents areamphoteric agents including, but not limited to, Lyso PC, CHAPS, andZwittergent. Alternatively, cationic agents including, but not limitedto, C16 TAB and Benzalkonium Chloride may be used as a lysis agent. Bothionic and non-ionic detergents are often used to break or lyse the cellwall or cell membrane components such as lipoproteins and glycoproteins.Common ionic detergents include, but are not limited to, SDS, Cholate,and Deoxycholate. Ionic detergents are good solubilizing agents.Antibodies retain their activity in 0.1% SDS or less. Common non-ionicdetergents include, but are not limited to, Octylglucoside, Digitonin,C12E8, Lubrol, Triton X-100, Noniodet P-40, Tween 20, and Tween 80.Non-ionic and mild ionic detergents are weaker denaturants and often areused to solubilize membrane proteins such as viral surface proteins.Additional lysis agents include, but are not limited to, urea andenzymes. Combinations of different lysis agents may be used to optimizethe lysing environment.

Surfactants are generally wetting agents and lower the surface tensionof a liquid. This then allows easier spreading by lowering theinterfacial tension between liquids. So, surfactants can interfere withthe natural binding of antigen and antibody or ligand and receptors. Theconcentrations are, therefore, experimentally chosen for each class oflysis agent. Once lysis occurs, it is important that the desired bindingreactions not be hindered. Generally, 0.001% lysis agent concentrationis considered the lower limit, and the upper limit is approximately 1%.There is an additive or synergistic effect when combinations of lysisagents are used. This expands the working range of concentration to runfrom approximately 0.001% to 1%. Finally, some undesirable non-specificbinding may be prevented at a Tween 20 concentration of 5%. In allcases, the total amount of lysis agent pre-loaded onto all locations ofan individual test strip must be sufficient to lyse barriers toimmunodetection, permitting practical operation of the test strip.

The lysis agent itself should not interfere with any other assaydetector or indicator agents and thus does not interfere with any otherassay interactions and reactions to such an extent as to preventpractical operation of the assay. A lysis agent should have sufficientshelf life to allow manufacture, distribution and storage before use ofa test strip in point-of-care testing.

In preferred embodiments where MxA is the viral marker, in situ lysisusing a combination of 1% to 6% weight/volume CHAPS and 0.5% to 2%weight/volume NP40 as the lysis agent is preferably used. As a morespecific example, 2 microliters of 100 mM HEPES buffer (pH 8.0)containing 5% CHAPS and 2% NP-40 with 150 mM Sodium Chloride, 0.1% BSA,and 0.1% Sodium Azide (all percentages weight/volume) are dried onto alysis zone of a test strip.

In a preferred embodiment, as shown in FIGS. 5A through 5D, the sampleis applied to the application zone (201) on a chromatography test strip(200). The sample passes a lysis zone (250), where a lysis agent willhave preferably been pre-loaded onto the test strip and is eluted by thetransport liquid. The lysis agent lyses any lysis-susceptible componentsin the sample in situ.

The chromatographic test strip contains a sample application zone (201),a lysis zone (250) containing a lysis agent, and a reagent zone (260)containing at least one labeled binding partner that binds to a viralmarker and at least one labeled binding partner that binds to abacterial marker that are eluted by and then able to migrate with asample transport liquid (e.g. a buffer solution). While the reagent zone(260) is shown downstream of the sample application zone in thesefigures, in alternative embodiments, the reagent zone (260) could beupstream of the sample application zone (see FIG. 4B), as long as thereagents encounter the sample at some point after the sample reaches thelysis zone and is effectively lysed. The labeled binding partners arecapable of specifically binding to a viral or bacterial marker ofinterest to form a complex which in turn is capable of specificallybinding to another specific reagent or binding partner in the detectionzone. Although not shown in these Figures, an absorbent pad, as well asother known lateral flow immunoassay components including, but notlimited to, a waste zone, a carrier backing, a housing, and an openingin the housing for result read out, may optionally also be a componentof the test strip (200) in these embodiments.

In a preferred embodiment, the lysis agent is localized in the lysiszone (250) between the sample application zone (201) and the reagentzone (260). The lysis agent is preferably soluble or miscible in thesample transport liquid, and the lysis agent is solubilized andactivated upon contact with the sample transport liquid. The sampletransport liquid then contains both lysis agent in solution orsuspension and sample components in suspension. Any lysis-susceptiblecomponents in a sample, then being exposed in suspension to the lysisagent, are themselves lysed in situ. The running buffer then carries thesample, including any lysis-freed components, to the detection zone(205).

The lysis zone (250) is preferably located between the sampleapplication zone (201) and the reagent zone (260), as shown in FIG. 5A.In other embodiments, the lysis zone (250) overlaps the sampleapplication zone (201), the reagent zone (260) or both the sampleapplication zone (201) and the reagent zone (260) as shown in FIGS. 5B,5C, and 5D, respectively. Note that the figures are schematic, and arenot drawn to scale. The amount of overlap between the different zones(as shown in FIGS. 5B through 5D) may be highly variable.

The test strip (200) also includes a detection zone (205) containing afirst section for detection of at least one bacterial marker, e.g. atest line (203), including an immobilized specific binding partner,complementary to the bacterial conjugate formed by the bacterial markerand its labeled binding partner. Thus, at the test line (203), detectionzone binding partners trap the bacterial labeled binding partners fromthe reagent zone (260) along with their bound bacterial markers. Thislocalization of the bacterial markers with their labeled bindingpartners gives rise to an indication at the test line (203). At the testline (203), the presence of a bacterial marker is determined byqualitative and/or quantitative readout of the test line (203)indication resulting from the accumulation of labeled binding partners.

The detection zone (205) also includes a second section for detection ofat least one viral marker, e.g. a test line (202), including animmobilized specific binding partner, complementary to the viralconjugate formed by the viral marker and its labeled binding partner.Thus, at the test line (202), detection zone binding partners trap theviral labeled binding partners from the reagent zone (260) along withtheir bound viral markers. This localization of the viral markers withtheir labeled binding partners gives rise to an indication at the testline (202). At the test line (202), the presence of a viral marker isdetermined by qualitative and/or quantitative readout of the test line(202) indication resulting from the accumulation of labeled bindingpartners. While test line (203) is upstream of test line (202) relativeto the direction of flow (208) in the figures, in alternativeembodiments, test line (202) is upstream of test line (203). In stillother embodiments, test lines (202) and (203) are located in the samelocation on the test strip.

Optionally, the detection zone (205) may contain further test lines todetect other bacterial and/or viral markers, as well as a control line(204). The control line (204) indicates that the labeled specificbinding partner traveled through the length of the assay, even though itmay not have bound any markers, thus confirming proper operation of theassay. As shown in FIGS. 5A through 5D, the control zone (204) ispreferably downstream of the test lines (203) and (202). However, inother embodiments, the control zone (204) may be located upstream ofeither or both of the test lines (203) and (202).

In a preferred embodiment, the control line (204) includes an antibodyor other recombinant protein which binds to a component of the elutionmedium or other composition being used in the test. In embodiments wherenucleic acids are the targets, the control line (204) preferablyincludes a nucleic acid complementary to the labeled nucleic acid beingused as a binding partner for the target nucleic acid.

Although only one test line is shown in the figures, multiple test linesare within the spirit of the invention. In some embodiments where thereare multiple targets, the presence of each target preferably correspondsto a separate test line (202). In other embodiments where there aremultiple targets, the presence of multiple targets may be indicated onthe same test line such that the presence of more than one target hasdifferent characteristics than the presence of a single target. Forexample, the presence of multiple targets on the same test line may bevisually indicated by a different color than the presence of each of thetargets alone.

In other embodiments, it is possible to have one or more mild lysisagents in the running buffer itself. In these embodiments, there is noadverse effect on the reagent zone which will be downstream and thesample can either be upstream or downstream of the reagent zone. Alysing enzyme in the running buffer can “target” its substrate and cutit to open up the cell membrane or cell wall. As an example, penicillincan excise or “punch a hole” in a susceptible bacteria. In otherembodiments, when the lysis agent is applied to the sample collectionmaterial, then the reagent zone may be upstream of the sampleapplication zone.

As an example, one or more lysis agents are dried onto the sampleapplication zone of a lateral flow strip. On a per strip basis, thelysis agent is made of approximately 2 microliters of 100 mM HEPESbuffer (pH 8.0) containing 5% CHAPS and 2% NP-40 with 150 mM SodiumChloride, 0.1% BSA, and 0.1% Sodium Azide (all percentagesweight/volume). Up to 10 microliters of whole blood are then added tothe sample application zone to be lysed in situ. MxA protein is releasedfrom inside white blood cells to react with an MxA monoclonal antibodyon a visual tag (colloidal gold or visible latex beads). This complextraverses with a running buffer containing Triton X-100 and is capturedby MxA monoclonal antibodies immobilized at the test line of thenitrocellulose membrane. This binding at the test line gives rise to avisible indication.

Accordingly, it is to be understood that the embodiments of theinvention herein described are merely illustrative of the application ofthe principles of the invention. Reference herein to details of theillustrated embodiments is not intended to limit the scope of theclaims, which themselves recite those features regarded as essential tothe invention.

What is claimed is:
 1. A method for determining if an infection isbacterial and/or viral, comprising the steps of: a) collecting a sample;b) transferring the sample to a sample analysis device comprising: areagent zone comprising: at least one first reagent specific to CRP suchthat, when CRP present in the sample contacts the first reagent, a firstlabeled complex forms; and at least one second reagent specific to MxAsuch that, when MxA present in the sample contacts the second reagent, asecond labeled complex forms; a detection zone comprising a firstbinding partner which binds to the first labeled complex and a secondbinding partner which binds to the second labeled complex; and a lysiszone comprising at least one lysis agent comprising approximately 1% to6% weight/volume CHAPS and approximately 0.5% to 2% weight/volume NP40;c) lysing the sample on the sample analysis device; and d) analyzing thesample for a presence of CRP and/or MxA.
 2. The method of claim 1,wherein step d) comprises the substeps of: i) eluting the sample on thesample analysis device; and ii) visually determining a result from thedetection zone.
 3. The method of claim 1, wherein a thresholdconcentration of CRP in a sample that elicits a positive result is atleast approximately 15 mg/L.
 4. The method of claim 1, wherein athreshold concentration of MxA in a sample that elicits a positiveresult is at least approximately 15 ng/ml.
 5. The method of claim 1,wherein the lysis agent comprises 100 mM HEPES buffer (pH 8.0) including5% CHAPS and 2% NP-40 with 150 mM Sodium Chloride, 0.1% BSA, and 0.1%Sodium Azide.
 6. The method of claim 1, wherein the sample comprises aplurality of leukocytes.
 7. The method of claim 6, wherein step c) lysesthe leukocytes.
 8. The method of claim 1, wherein the sample analysisdevice is a lateral flow chromatography test strip.
 9. The method ofclaim 1, wherein the sample is a sample of whole blood.
 10. The methodof claim 1, wherein the presence of MxA or CRP is indicated by testlines located in the detection zone visible to the naked eye.
 11. Themethod of claim 10, wherein the presence of MxA is indicated by a firsttest line located in the detection zone and the presence of CRP isindicated by a second test line located in the detection zone.
 12. Themethod of claim 11, wherein the first test line displays a first colorwhen positive and the second test line displays a second color differentfrom the first color when positive.
 13. The method of claim 12, whereinboth the first test line and the second test line are located in thesame space on the sample analysis device such that a third color isformed when both the first test line and the second test line arepositive.
 14. The method of claim 11, wherein the first test line isspatially separate from the second test line on the sample analysisdevice.
 15. The method of claim 1, wherein the sample is a blood sample.16. The method of claim 1, wherein the sample comprises leukocytes. 17.The method of claim 1, wherein the sample is taken from a locationselected from the group consisting of: peripheral blood, nasopharyngealaspirates, tears, spinal fluid, and middle ear aspirates.